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Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands.
Biochemistry. 1994 Jun 07; 33(22):6925-35.B

Abstract

The covalently attached heme and its axial ligands not only are essential for the structure and function of cytochrome c but they also play an important role in the folding process. Under typical denaturing conditions (concentrated guanidine hydrochloride or urea near pH 7), one of the axial ligands, His 18, remains bound to the oxidized heme iron, but the second ligand, Met 80, is replaced by a non-native histidine ligand (His 26 or His 33 in horse cytochrome c). Using quenched-flow and NMR methods, hydrogen exchange rates were measured for several individual amide protons in guanidine-denatured horse cytochrome c. The observation of a single highly protected (140-fold) backbone amide, that of His 18, suggests the presence of a persistent H-bond consistent with heme ligation of the His 18 side chain in the unfolded state. Heme absorbance changes induced by rapid acidification of oxidized cytochrome c in 4.5 M guanidine hydrochloride from pH 7.8 to 4.6 or below exhibit two kinetic phases with rates of 110 and 25 s-1, attributed to the dissociation of non-native histidine ligands from the heme in the unfolded state. The kinetics of folding from guanidine-denatured cytochrome c under a variety of initial and final conditions was investigated by stopped-flow methods, using tryptophan fluorescence as a conformational probe and Soret absorbance as a probe for the ligation state of the heme. A fast kinetic phase (80 s-1) accompanied by a major decrease in fluorescence and a minor absorbance change coincides with the formation of a partially folded intermediate with interacting chain termini detected in earlier pulsed NH exchange measurements [Roder, H., Elöve, G. A., & Englander, S. W. (1988) Nature 335, 700]. At neutral pH, an intermediate kinetic phase (1.8 s-1) accounts for 78% of the absorbance change and 47% of the fluorescence change. In contrast, the folding kinetics at pH 5 is dominated by the fast phase, and the amplitude of the intermediate phase is reduced to approximately 10%. The pH-dependent amplitude changes show titration behavior with an apparent pK of approximately 5.7, consistent with the protonation of a single histidine residue. The intermediate phase can also be suppressed by the addition of 20 mM imidazole. Since both of these conditions interfere with histidine ligation, the intermediate kinetic phase is attributed to the presence of a non-native histidine ligand (His 26 or His 33) that can become trapped in a partially folded intermediate.(

ABSTRACT

TRUNCATED AT 400 WORDS)

Authors+Show Affiliations

Institute for Cancer Research, Philadelphia, Pennsylvania 19111.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8204626

Citation

Elöve, G A., et al. "Kinetic Mechanism of Cytochrome C Folding: Involvement of the Heme and Its Ligands." Biochemistry, vol. 33, no. 22, 1994, pp. 6925-35.
Elöve GA, Bhuyan AK, Roder H. Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. Biochemistry. 1994;33(22):6925-35.
Elöve, G. A., Bhuyan, A. K., & Roder, H. (1994). Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. Biochemistry, 33(22), 6925-35.
Elöve GA, Bhuyan AK, Roder H. Kinetic Mechanism of Cytochrome C Folding: Involvement of the Heme and Its Ligands. Biochemistry. 1994 Jun 7;33(22):6925-35. PubMed PMID: 8204626.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. AU - Elöve,G A, AU - Bhuyan,A K, AU - Roder,H, PY - 1994/6/7/pubmed PY - 1994/6/7/medline PY - 1994/6/7/entrez SP - 6925 EP - 35 JF - Biochemistry JO - Biochemistry VL - 33 IS - 22 N2 - The covalently attached heme and its axial ligands not only are essential for the structure and function of cytochrome c but they also play an important role in the folding process. Under typical denaturing conditions (concentrated guanidine hydrochloride or urea near pH 7), one of the axial ligands, His 18, remains bound to the oxidized heme iron, but the second ligand, Met 80, is replaced by a non-native histidine ligand (His 26 or His 33 in horse cytochrome c). Using quenched-flow and NMR methods, hydrogen exchange rates were measured for several individual amide protons in guanidine-denatured horse cytochrome c. The observation of a single highly protected (140-fold) backbone amide, that of His 18, suggests the presence of a persistent H-bond consistent with heme ligation of the His 18 side chain in the unfolded state. Heme absorbance changes induced by rapid acidification of oxidized cytochrome c in 4.5 M guanidine hydrochloride from pH 7.8 to 4.6 or below exhibit two kinetic phases with rates of 110 and 25 s-1, attributed to the dissociation of non-native histidine ligands from the heme in the unfolded state. The kinetics of folding from guanidine-denatured cytochrome c under a variety of initial and final conditions was investigated by stopped-flow methods, using tryptophan fluorescence as a conformational probe and Soret absorbance as a probe for the ligation state of the heme. A fast kinetic phase (80 s-1) accompanied by a major decrease in fluorescence and a minor absorbance change coincides with the formation of a partially folded intermediate with interacting chain termini detected in earlier pulsed NH exchange measurements [Roder, H., Elöve, G. A., & Englander, S. W. (1988) Nature 335, 700]. At neutral pH, an intermediate kinetic phase (1.8 s-1) accounts for 78% of the absorbance change and 47% of the fluorescence change. In contrast, the folding kinetics at pH 5 is dominated by the fast phase, and the amplitude of the intermediate phase is reduced to approximately 10%. The pH-dependent amplitude changes show titration behavior with an apparent pK of approximately 5.7, consistent with the protonation of a single histidine residue. The intermediate phase can also be suppressed by the addition of 20 mM imidazole. Since both of these conditions interfere with histidine ligation, the intermediate kinetic phase is attributed to the presence of a non-native histidine ligand (His 26 or His 33) that can become trapped in a partially folded intermediate.(ABSTRACT TRUNCATED AT 400 WORDS) SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8204626/Kinetic_mechanism_of_cytochrome_c_folding:_involvement_of_the_heme_and_its_ligands_ L2 - https://antibodies.cancer.gov/detail/CPTC-MET-1 DB - PRIME DP - Unbound Medicine ER -